Mitochondrial DNA Deletion Between About Residues 12317-16254 for Use in the Detection of Cancer

ABSTRACT

The present invention relates to methods for predicting, diagnosing and monitoring cancer. The methods comprise obtaining biological samples, extracting mitochondrial DNA (mtDNA) from the samples, quantifying mitochondrial DNA mutation in the sample and comparing the level of mtDNA mutation with a reference value. The methods of the invention may also be effective in screening for new therapeutic agents and treatment regimes, and may also be useful for monitoring the response of a subject to a preventative or therapeutic treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Application No. 61/002,637,filed Nov. 9, 2007, the entire contents of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention pertains to the field of mitochondrial genomics.In particular it is related to the detection of human mitochondrialgenome mutations and their utility as an indicators of cancer.

BACKGROUND OF THE INVENTION Mitochondrial DNA as a Diagnostic Tool

Mitochondrial DNA (mtDNA) sequence dynamics are important diagnostictools. Mutations in mtDNA are often preliminary indicators of developingdisease, often associated with nuclear mutations, and act as biomarkersspecifically related to: disease, such as but not limited to, tissuedamage and cancer from smoking and exposure to second hand tobacco smoke(Lee et al., 1998; Wei, 1998); longevity, based on accumulation ofmitochondrial genome mutations beginning around 20 years of age andincreasing thereafter (von Wurmb, 1998); metastatic disease caused bymutation or exposure to carcinogens, mutagens, ultraviolet radiation(Birch-Machin, 2000); osteoarthritis; cardiovascular, Alzheimer,Parkinson disease (Shoffner et al., 1993; Sherratt et al., 1997; Zhanget al, 1998); age associated hearing loss (Seidman et al., 1997); opticnerve degeneration and cardiac dysrhythmia (Brown et al., 1997; Wallaceet al., 1988); chronic progressive external exopthalmoplegia (Taniike etal., 1992); atherosclerosis (Bogliolo et al., 1999); papillary thyroidcarcinomas and thyroid tumours (Yeh et al., 2000); as well as others(e.g. Naviaux, 1997; Chinnery and Turnbull, 1999).

Mutations at specific sites of the mitochondrial genome can beassociated with certain diseases. For example, mutations at positions4216, 4217 and 4917 are associated with Leber's Hereditary OpticNeuropathy (LHON) (Mitochondrial Research Society; Huoponen (2001);MitoMap). A mutation at 15452 was found in 5/5 patients to be associatedwith ubiquinol cytochrome c reductase (complex III) deficiency (Valnotet al. 1999).

Specifically, these mutations or alterations include point mutations(transitions, transversions), deletions (one base to thousands ofbases), inversions, duplications, (one base to thousands of bases),recombinations and insertions (one base to thousands of bases). Inaddition, specific base pair alterations, deletions, or combinationsthereof have been found to be associated with early onset of prostate,skin, and lung cancer, as well as aging (e.g. Polyak et al., 1998),premature aging, exposure to carcinogens (Lee et al., 1998), etc.

Prostate Cancer

Prostate cancer is a frequently diagnosed solid tumour that most likelyoriginates in the prostate epithelium (Huang et al. 1999). In 1997,nearly 10 million American men were screened for prostate specificantigen (PSA), the presence of which suggests prostate cancer (Woodwell,1999). Indeed, this indicates an even higher number of men screened byan initial digital rectal exam (DRE). In the same year, 31 million menhad a DRE (Woodwell, 1999). Moreover, the annual number of newlydiagnosed cases of prostate cancer in the United States is estimated at179,000 (Landis et al., 1999). It is the second most commonly diagnosedcancer and second leading cause of cancer mortality in Canadian men. In1997 prostate cancer accounted for 19,800 of newly diagnosed cancers inCanadian men (28%) (National Cancer Institute of Canada). It isestimated that 30% to 40% of all men over the age of forty-nine (49)have some cancerous prostate cells, yet only 20% to 25% of these menhave a clinically significant form of prostate cancer (SpringNet—CEConnection, internet, www.springnet.com/ce/j803a.htm). Prostate cancerexhibits a wide variety of histological behaviour involving bothendogenous and exogenous factors, i.e. socio-economic situations, diet,geography, hormonal imbalance, family history and genetic constitution(Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNAalterations have been previously associated with prostate cancer, theneed exists for further markers for the detection of prostate cancer.

Breast Cancer

Breast cancer is a cancer of the glandular breast tissue and is thefifth most common cause of cancer death. In 2005, breast cancer caused502,000 deaths (7% of cancer deaths; almost 1% of all deaths) worldwide(World Health Organization Cancer Fact Sheet No. 297). Among womenworldwide, breast cancer is the most common cancer and the most commoncause of cancer death (World Health Organization Cancer Fact Sheet No.297). Although certain mtDNA alterations have been previously associatedwith breast cancer, for example in Parrella et al. (Cancer Research: 61,2001), the need exists for further markers for the detection of breastcancer.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

The present invention pertains to mitochondrial DNA mutations for use inthe detection of cancer. In accordance with an aspect of the presentinvention, there is provided a method of detecting a cancer in anindividual comprising:

-   -   a) obtaining a biological sample from the individual;    -   b) extracting mitochondrial DNA (mtDNA) from the sample;    -   c) quantifying the amount of mtDNA in the sample having a        deletion in the mtDNA sequence between about residue 12317 and        about residue 16254 of the human mtDNA genome; and    -   d) comparing the amount of mtDNA in the sample having the        deletion to at least one known reference value.

In accordance with another aspect of the present invention, there isprovided a method of monitoring an individual for the development of acancer comprising:

-   -   a) obtaining a biological sample;    -   b) extracting mitochondrial DNA (mtDNA) from the sample;    -   c) quantifying the amount of mtDNA in the sample having a        deletion in the mtDNA sequence between about residue 12317 and        about residue 16254 of the human mtDNA genome; and    -   d) repeating steps a) to c) over a duration of time;

wherein an increasing level of the deletion over the duration of time isindicative of cancer.

In accordance with another aspect of the present invention, there isprovided a method of detecting a cancer in an individual comprising:

-   -   a) obtaining a biological sample from the individual;    -   b) extracting mitochondrial DNA (mtDNA) from the sample;    -   c) quantifying the amount of mtDNA in the sample having a        sequence corresponding to the sequence as set forth in SEQ ID        NO: 1 or SEQ ID NO: 2; and    -   d) comparing the amount of mtDNA in the sample corresponding to        SEQ ID NO: 1 or SEQ ID NO: 2 to at least one known reference        value.

In accordance with another aspect of the present invention, there isprovided a diagnostic kit for carrying out the method of the inventioncomprising:

-   -   (a) material for collecting one or more biological samples; and    -   (b) suitable primers and reagents for detecting the mtDNA        deletion.

BRIEF DESCRIPTION OF THE FIGURES

These and other features of the invention will become more apparent inthe following detailed description in which reference is made to theappended drawings.

FIG. 1 is a graph showing cycle threshold as related to Example 1.

FIG. 2 shows a ROC curve illustrating the specificity and sensitivity ofone embodiment of the present invention.

FIG. 3 is a graph showing cycle threshold as related to Example 2.

FIG. 4 shows a ROC curve illustrating the specificity and sensitivity ofanother embodiment of the present invention.

FIG. 5 is a schematic diagram showing the design and sequence of aprimer useful for the detection of the 4 kb deletion.

FIG. 6 shows a ROC curve illustrating the specificity and sensitivity ofanother embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of predicting, diagnosing andmonitoring cancer. The methods comprise obtaining one or more biologicalsamples, extracting mitochondrial DNA (mtDNA) from the samples,quantifying the amount of a mitochondrial mutation in the samples andcomparing the quantity of the mutation in a sample with a referencevalue. In this regard, the methods provide a comprehensive tool fordetermining disease onset and for assessing the predisposition of anindividual to cancer. The methods also allow for the monitoring of anindividual's risk factors over time and/or for monitoring a patient'sresponse to therapeutic agents and treatment regimes.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term “about” refers to an understood variation fromthe stated value. It is to be understood that such a variation is alwaysincluded in any given value provided herein, whether or not it isspecifically referred to.

As defined herein, “biological sample” refers to a tissue or bodilyfluid containing cells from which mtDNA can be obtained. For example,the biological sample can be derived from tissue such as breast orprostate tissue, or from blood, saliva, cerebral spinal fluid, sputa,urine, mucous, synovial fluid, peritoneal fluid, amniotic fluid and thelike. The biological sample may be a surgical specimen or a biopsyspecimen. The biological sample can be used either directly as obtainedfrom the source or following a pre-treatment to modify the character ofthe sample. Thus, the biological sample can be pre-treated prior to useby, for example, preparing plasma or serum from blood, disrupting cells,preparing liquids from solid materials, diluting viscous fluids,filtering liquids, distilling liquids, concentrating liquids,inactivating interfering components, adding reagents, and the like.

As used herein, “cycle threshold” (C_(T)) is the point at which targetamplification using real-time PCR rises above background, as indicatedby a signal such as a fluorescence signal. The C_(T) is inverselyrelated to the quantity of the sequence being investigated.

As used herein, “diagnostic” or “diagnosing” means using the presence orabsence of a mutation or combination of mutations as a factor in diseasediagnosis or management. The detection of the mutation(s) can be a stepin the diagnosis of a disease.

As used herein, “deletion” means removal of a region of mtDNA from acontiguous sequence of mtDNA. Deletions can range in size from one baseto thousands of bases or larger.

As used herein, “mitochondrial DNA” or “mtDNA” is DNA present inmitochondria.

As used herein, “mutation” encompasses any modification or change inmitochondrial DNA from the wild type sequence, including withoutlimitation point mutations, transitions, insertions, transversions,translocations, deletions, inversions, duplications, recombinations orcombinations thereof. The modification or change of the sequence canextend from a single base change to the addition or elimination of anentire DNA fragment.

As defined herein, “sensitivity” refers to the fraction of truepositives (true positive rate) results obtained using the method of thepresent invention.

As defined herein, “specificity” refers to the fraction of falsepositives (false positive rate) results obtained using the method of thepresent invention.

The terms “therapy” and “treatment,” as used interchangeably herein,refer to an intervention performed with the intention of improving asubject's status. The improvement can be subjective or objective and isrelated to ameliorating the symptoms associated with, preventing thedevelopment of, or altering the pathology of a disease. Thus, the termstherapy and treatment are used in the broadest sense, and include theprevention (prophylaxis), moderation, reduction, and curing of adisease, at various stages. Preventing deterioration of a subject'sstatus is also encompassed by the term. Subjects in need oftherapy/treatment thus include those already having the disease, as wellas those prone to, or at risk of developing, the disease, and those inwhom the disease is to be prevented.

Assays for Predicting, Diagnosing and Monitoring Cancer Assay forDetection of Mitochondrial Mutation

Mitochondrial DNA (mtDNA) dynamics are an important diagnostic tool.

Mutations in mtDNA are often preliminary indicators of developingdisease and may act as biomarkers indicative of risk factors associatedwith disease onset. As discussed herein, measuring the level ofmitochondrial DNA aberration in a biological sample can determine thepresence of one or more cancers and identify the potential risk orpredisposition of a patient to one or more cancers. Furthermore,measurement of mtDNA at regular intervals can provide health careprofessionals with a real-time, quantitative monitoring tool formeasuring the progression of a patient over time and/or as an assessmentfor treatment recommendations in order to determine their effectivenessin preventing or treating cancer.

The present invention, therefore, provides methods for predicting,diagnosing or monitoring cancer, comprising obtaining one or morebiological samples, extracting mitochondrial DNA (mtDNA) from thesamples, and assaying the samples for mitochondrial mutation by:quantifying the amount of an mtDNA aberration in the sample andcomparing the level of the aberration with a reference value. As wouldbe understood by those of skill in the art, the reference value is basedon whether the method seeks to predict, diagnose or monitor cancer.Accordingly, the reference value may relate to mtDNA data collected fromone or more known non-cancerous biological samples, from one or moreknown cancerous biological samples, and/or from one or more biologicalsamples taken over time. These reference values are used for comparisonwith the mtDNA data collected from the one or more biological sampleswherein, for example, a similar or elevated amount of deletion in thebiological sample compared to the reference sample is indicative of apredisposition to or the onset of cancer, or wherein an increasing levelof the deletion over time is indicative of cancer onset.

In accordance with an aspect of the invention, the methods forpredicting, monitoring and diagnosing cancer comprise an assay fordetecting and quantifying one or more mitochondrial mutations. Inaccordance with one embodiment of the invention, the mutation is anmtDNA deletion. In accordance with another embodiment, the mutation isan mtDNA deletion of 3926 bp of mtDNA (referred to herein as “the 4 kbdeletion” or “4 kb sequence”). In accordance with yet anotherembodiment, the mutation is an mtDNA deletion having the sequence as setforth in SEQ ID NO:1 or SEQ ID NO:2, there being no difference betweenSEQ ID NO: 1 and SEQ ID NO: 2 when in circular form.

The 4 kb deletion spans approximately nucleotides 12317 and 16254 of thehuman mtDNA genome. The human mtDNA genome is listed herein as SEQ IDNO:3 (Genbank accession no. AC_(—)000021). The 4 kb deletion ischaracterized by direct flanking repeats 12 by in size, with the repeatslocated at positions 12317-12328 and 16243 to 16254. The repeat sequenceis 5′-TGCAACTCCAAA-3′. Thus, in accordance with one embodiment of theinvention, the mutation is an mtDNA deletion of between about residue12317 and about residue 16254 of the human mtDNA genome.

The inventors have determined, as provided by way of example below, thatthis deletion is associated with cancer and in particular prostate andbreast cancer. Therefore, such deletion provides an accurate biomarkerand, therefore, a valuable tool for the detection, diagnosis, ormonitoring of cancer in at least these tissues.

The deletion results in the creation of two deletion monomers, one of 4kb in size (small sublimon) and one of approximately 12.5 kb in size(large sublimon). The occurrence of the deletion may be detected byeither identifying the presence of the small sublimon or the largesublimon, the 4 kb or 12.5 kb sequence respectively.

Exemplary methods for assaying the mitochondrial mutation are providedin the Example section. Extraction of mtDNA from a sample may beundertaken using any suitable known method. MtDNA extraction is followedby amplification of all or a region of the mitochondrial genome, and mayinclude sequencing of the mitochondrial genome, as is known in the artand described, for example, in Current Protocols in Molecular Biology(Ausubel et al., John Wiley & Sons, New York, 2007). Likewise, methodsfor detecting the presence of mutations in the mtDNA can be selectedfrom suitable techniques known to those skilled in the art. For example,analyzing mtDNA can comprise sequencing the mtDNA, amplifying mtDNA byPCR, Southern, Northern, Western South-Western blot hybridizations,denaturing HPLC, hybridization to microarrays, biochips or gene chips,molecular marker analysis, biosensors, melting temperature profiling ora combination of any of the above.

Any suitable means to sequence mitochondrial DNA may be used.Preferably, mtDNA is amplified by PCR prior to sequencing. The method ofPCR is well known in the art and may be performed as described in Mullisand Faloona, 1987, Methods Enzymol., 155: 335. PCR products can besequenced directly or cloned into a vector which is then placed into abacterial host. Examples of DNA sequencing methods are found in Brumley,R. L. Jr. and Smith, L. M., 1991, Rapid DNA sequencing by horizontalultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 andLuckey, J. A., et al, 1993, High speed DNA sequencing by capillary gelelectrophoresis, Methods Enzymol. 218: 154-172. The combined use of PCRand sequencing of mtDNA is described in Hopgood, R., et al, 1992,Strategies for automated sequencing of human mtDNA directly from PCRproducts, Biotechniques 13:82-92 and Tanaka, M. et al, 1996, Automatedsequencing of mtDNA, Methods Enzymol. 264:407-421.

Although real-time quantitative PCR methods, as described in theexamples below, represent the preferred means for detecting andquantifying the presence or absence of the 4 kb deletion, other methodswould be well known to an individual of skill in the art and could beutilized as indicated above. In addition, quantification of the deletioncould be made using Bio-Rad's Bioplex™ System and Suspension Arraytechnology. Generally, the method requires amplification andquantification of sequences using any known methods.

The following primer sequences are examples of primers that may be usedfor the detection of the 4 kb deletion:

4 forward (binds to bases 12313-12328/16255-16267 of the human mtDNAgenome) 5′-TTGGTGCAACTCCAAAGCCACCCCTCACC-3′ (SEQ ID NO: 4);

4 reverse (binds to bases 16391-16409 of the human mtDNA genome)5′-AGGATGGTGGTCAAGGGAC-3′ (SEQ ID NO: 5).

In one embodiment of the present invention, a pair of amplificationprimers are used to amplify a target region indicative of the presenceof the 4 kb deletion. In this embodiment, one of the pair ofamplification primers overlaps a spliced region of mtDNA after deletionof the 4 kb sequence has occurred and the mtDNA has reformed as acircular mtDNA molecule (eg. a splice at a position between 12328 and16255 of the mtDNA genome). Therefore, extension of the overlappingprimer can only occur if the 4 kb section is deleted. FIG. 5 is aschematic diagram showing the design and sequence of the primer (ie. SEQID NO: 4).

In another embodiment of the present invention, a pair of amplificationprimers are used to amplify a target region associated with the deleted4 kb sequence. The deleted 4 kb sequence, upon deletion, may reform as acircular mtDNA molecule. In this embodiment, one of the pair ofamplification primers overlaps the rejoining site of the ends of the 4kb sequence. Thus, an increase in the amount of the 4 kb moleculedetected in a sample is indicative of cancer.

In still another embodiment of the present invention, the breakpoint ofthe deletion is unknown thereby resulting in two possibilities forprimer location. In this embodiment, two separate forward primers may bedesigned to amplify the target region associated with the deleted 4 kbsequence. The following primer sequences are examples of those that maybe used for the detection of the 4 kb deletion in this scenario:

Forward Primers:

Primer A (binds to bases 12313-12328/16255-16267 of the human mtDNAgenome) 5′-TTGGTGCAACTCCAAAGCCACCCCTCACC-3′ (SEQ ID NO: 4);

Primer B (binds to bases 12302-12316 of the human mtDNA genome)5′-CCCAAAAATTTTGGTGCAACTCCAAAGCCAC-3′ (SEQ ID NO: 6).

Reverse Primer:

Primer C (binds to bases 16391-16409 of the human mtDNA genome)5′-AGGATGGTGGTCAAGGGAC-3′ (SEQ ID NO: 5).

As would be understood by a person of skill in the art, the forwardprimers A or B can be used with reverse primer C to create PCR productsthat are useful in qPCR assays.

Biological Sample

The present invention provides for diagnostic tests which involveobtaining or collecting one or more biological samples. In the contextof the present invention, “biological sample” refers to a tissue orbodily fluid containing cells from which mtDNA can be obtained. Forexample, the biological sample can be derived from tissue including, butnot limited to, breast, prostate, nervous, muscle, heart, stomach, colontissue and the like; or from blood, saliva, cerebral spinal fluid,sputa, urine, mucous, synovial fluid, peritoneal fluid, amniotic fluidand the like. The biological sample may be obtained from a cancerous ornon-cancerous tissue and may be a surgical specimen or a biopsyspecimen.

The biological sample can be used either directly as obtained from thesource or following a pre-treatment to modify the character of thesample. Thus, the biological sample can be pre-treated prior to use by,for example, preparing plasma or serum from blood, disrupting cells,preparing liquids from solid materials, diluting viscous fluids,filtering liquids, distilling liquids, concentrating liquids,inactivating interfering components, adding reagents, and the like.

One skilled in the art will understand that more than one sample typemay be assayed at a single time (i.e. for the detection of more than onecancer). Furthermore, where a course of collections are required, forexample, for the monitoring of risk factors or cancer over time, a givensample may be diagnosed alone or together with other sample takenthroughout the test period. In this regard, biological samples may betaken once only, or at regular intervals such as biweekly, monthly,semi-annually or annually.

One of skill will also appreciate that mitochondrial DNA targets are inmuch greater abundance (approximately 1000 fold greater) than nucleicacid targets and as such sample sizes comprising extremely low yields ofnucleic acids would be suitable for use with the present invention.

Applications for Predicating, Diagnosing and Monitoring CancerDiagnosing and Monitoring Cancer

The prevalence of cancer in most tissue types and age groupsnecessitates the availability of a tool to not only detect the presenceof cancer, but also to monitor the success and appropriateness ofpreventative measures and therapies being advised to prevent onset,progression and spread of the disease. Measuring the level ofmitochondrial DNA deletions in one or more biological samples of anindividual can provide initial diagnosis of risk factors, cancer and/orstages of the disease.

The system and method of the present invention, for example, may be usedto detect cancer at an early stage, and before any histologicalabnormalities. Furthermore, sample testing at regular intervals such asbiweekly, monthly, semi-annually or annually (or any other suitableinterval) can provide health care professionals with a real-time,quantitative monitoring tool to compare against treatmentrecommendations to determine their effectiveness in preventing ortreating the disease.

Turning now to the examples, in one embodiment the present invention maybe used for detecting the presence of pre-neoplasia, neoplasia andprogression towards potential malignancy of prostate cancer and breastcancer. In one aspect, the present invention involves the detection andquantification of the 4 kb mtDNA deletion for the detection, diagnosis,and/or monitoring of cancer. In this method, mtDNA is extracted from abiological sample (for example body tissue, or body fluids such asurine, prostate massage fluid). The extracted mtDNA is then tested inorder to determine the levels (ie. quantity) of the 4 kb deletion in thesample. In tests conducted by the present inventors, the levels of thedeletion were found to be elevated in samples obtained from subjectswith cancer when compared to samples obtained from subjects withoutcancer. Based on the information and data supplied below, the inventorshave concluded that elevated levels of the 4 kb deletion in human mtDNAis indicative of cancer.

In another embodiment, samples of, for instance prostate tissue,prostate massage fluid, urine or breast tissue, are obtained from anindividual and tested over a period of time (eg. years) in order tomonitor the genesis or progression of cancer. Increasing levels of the 4kb deletion over time could be indicative of the beginning orprogression of cancer.

One of ordinary skill in the art will appreciate that analysing one ormore biological samples from an individual for quantification of amitochondrial DNA target provides a means for a health care worker tomonitor the effectiveness of treatment regimes. One of ordinary skillwill also appreciate the utility of mtDNA analysis for use by healthcare providers in identifying (and providing recommendations for)lifestyle habits, such as poor diet and exercise, or activities thatcause over exposure of an individual to known carcinogens (eg. tobacco,pollutants).

Another aspect of the invention provides methods for confirming orrefuting the results of a cancer biopsy test from a biopsy sample (eg.prostate or breast cancer), comprising: obtaining non-cancerous tissuefrom a biopsy sample; and detecting and quantifying the amount of the 4kb mtDNA deletion in the non-diseased tissue.

Determining Genetic Predisposition to Cancer

In order to fully evaluate an individual's risk of one or more cancersit is imperative that health care providers are provided with as muchinformation as possible to understand and communicate their patient'srisk factors. The utilization of the present invention to determine thelevel of mtDNA aberration will not only prove helpful in assessing anindividual's susceptibility to one or more cancers, it provides avaluable tool to identify patients with greater risk who are potentiallyin need of more aggressive monitoring and treatment measures.

In this regard, the various examples provided below illustrate adifference in the amount of mtDNA having the 4 kb deletion betweensamples obtained from subjects having cancer, and subjects withoutcancer. The amount of the 4 kb deletion was found to be higher in thesamples obtained from subjects having cancer. This determination wasmade by comparing the amount of the 4 kb deletion in the samples fromknown cancer cells and/or known non-cancer cells.

As such, the inventors determined that screening of biological sampleswould prove useful in identifying an individual's predisposition to oneor more cancers. Thus, in accordance with one embodiment of the presentinvention there is provided a method for screening individuals forcancer from one or more biological samples comprising: obtaining the oneor more samples, and detecting and quantifying the level of the 4 kbmtDNA deletion in the samples. In a specific embodiment of theinvention, there is provided a method for screening individuals forprostate or breast cancer from a body fluid or tissue sample comprising;obtaining the body fluid or tissue sample, and detecting and quantifyingthe level of the 4 kb mtDNA deletion in the body fluid or tissue sample.

Age related accumulation of the 4 kb mtDNA deletion may also predisposean individual to, for example, prostate cancer or breast cancer, whichis prevalent in middle aged and older men, and middle aged and olderwomen, respectively. Similarly, an accumulation of the 4 kb mtDNAdeletion may be associated with a particular lifestyle based on anindividual's diet, exercise habits, and exposure to known carcinogens.Thus, in accordance with one aspect of the invention, a method isprovided wherein regular cancer screening may take place by monitoringover time the amount of the 4 kb deletion in one or more biologicalsamples, non-limiting examples of which include breast and prostatetissues or body fluids such as prostate massage fluid, or urine.

Evaluation of Therapeutic Agents

The method of the present invention may also be used for screeningpotential therapeutic agents for use in cancer treatment or formonitoring the therapeutic effect of such agents. The method of thepresent invention may be used to measure various biomarkers associatedwith the cancers identified herein. The ability to assess the level ofDNA damage in any biological sample at any time point provides thefoundation for a unique and informative screening test for anindividual's health and to assess the safety and efficacy of existingand new therapeutic agents and treatment regimes. Furthermore, byidentifying the specific genetic changes underlying a subject's state ofhealth, it may be readily determined whether and to what extent apatient will respond to a particular therapeutic agent or regime.

Kits

The present invention provides diagnostic/screening kits for use in aclinical environment. Such kits could not only include one or moresampling means, but other materials necessary for the identification ofmtDNA mutations.

The kits can optionally include reagents required to conduct adiagnostic assay, such as buffers, salts, detection reagents, and thelike. Other components, such as buffers and solutions for the isolationand/or treatment of a biological sample, may also be included in thekit. One or more of the components of the kit may be lyophilised and thekit may further comprise reagents suitable for the reconstitution of thelyophilised components.

Where appropriate, the kit may also contain reaction vessels, mixingvessels and other components that facilitate the preparation of the testsample. The kit may also optionally include instructions for use, whichmay be provided in paper form or in computer-readable form, such as adisc, CD, DVD or the like.

In one aspect of the invention there is provided a kit for diagnosingcancer comprising means for extraction of mtDNA, primers, reagents andinstructions.

In another aspect of the invention there is provided a kit fordiagnosing cancer, for example prostate or breast cancer, comprisingmeans for extraction of mtDNA, primers having the nucleic acid sequencesrecited in SEQ ID NOs: 4 and 5, reagents and instructions.

In another aspect of the invention there is provided a kit fordiagnosing cancer, for example prostate or breast cancer, comprisingmeans for extraction of mtDNA, primers having the nucleic acid sequencesrecited in SEQ ID NOs: 6 and 5, reagents and instructions.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It will be understood that theseexamples are intended to describe illustrative embodiments of theinvention and are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 Association of Prostate Cancer with 4 kb Deletion inHuman mtDNA

Urine samples were collected from five patients who had been diagnosedwith prostate cancer and five who had a needle biopsy procedure whichwas unable to detect prostate malignancy. These samples were collectedfollowing a digital rectal exam (DRE) to facilitate the collection ofprostate cells.

Upon receipt of the samples a 5 ml aliquot was removed and then 2 mlswere centrifuged at 14,000×g to form a pellet. The supernatant wasremoved and discarded.

Pellets were resuspended in 200 ul phosphate buffered saline solution.Both the resuspended pellet and the whole urine sample were subjected toa DNA extraction procedure using the QiaAMP DNA Mini Kit (Qiagen P/N51304) according to the manufacturer's directions. The resulting DNAextracts were then quantified using a NanoDrop ND-1000 Spectrophotometerand normalized to a concentration of 0.1 ng/ul.

Samples were analyzed by quantitative real-time PCR with the 4 kbdeletion specific primers according to the following:

1× iQ SYBR Green Supermix (Bio-Rad product no. 170-8880)

100 nmol forward primer (5′-TTGGTGCAACTCCAAAGCCACCCCTCACC-3′) (SEQ IDNO: 4)

100 nmol reverse primer (5′-AGGATGGTGGTCAAGGGAC-3′) (SEQ ID NO: 5)

1 ng template DNA in a 25 ul reaction

Reactions were cycled on an Opticon 2 DNA Engine (Bio-Rad Canada)according to the following protocol:

-   -   1. 95° C. for 3 minutes    -   2. 95° C. for 30 seconds    -   3. 69° C. for 30 seconds    -   4. 72° C. for 30 seconds    -   5. Plate Read    -   6. Repeat steps 2-5 44 times    -   7. 72° C. for 10 minutes    -   8. Melting Curve from 50° C. to 105° C., read every 1° C., hold        for 3 seconds    -   9. 10° C. Hold

Results

Results from the urine pellet did not yield significant differences inthe mean cycle threshold observed or a useful cutoff point. However, theresults from the whole urine sample did yield significant differences asprovided below.

Tables 1 and 2, and FIG. 1 show the difference in the mean C_(T) scoresfor urine samples from subjects having prostate malignant tissue andbenign tissue at the 0.04 significance level.

TABLE 1 Mean Values for C_(T) scores: Urine Samples Std. Error N MeanStd. Deviation Mean Benign 7 38.0357 3.40974 1.288876 Malignant 731.9300 6.12583 2.31534

TABLE 2 Significance Test for Mean C_(T) scores Independent Samples TestLevene's Test for Equality Means Test for 95% Confidence Equality ofSig. Std. Interval of the CTt40 Variances (2- Mean Error Differencefluid F Sig. t df tailed) Diff. Diff. Lower Upper Equal 1.707 .216 230412 .040 610571 264985 .33218 11.87925 variances assumed Equal 2304 9392.046 610571 264985 .14927 12.06215 variances not assumed

Tables 3 and 4, and FIG. 2 illustrate that when using a cut-off cyclethreshold of 36.255 the sensitivity of the assay for prostate cancer is86% and the specificity is 86%.

FIG. 2 is a Receiver Operating Characteristic (ROC) curve illustratingthe specificity and sensitivity of the 4 kb mtDNA deletion as a markerfor prostate cancer when testing urine. These results were obtainedusing a cutoff C_(T) of 36.255. The sensitivity of the marker at thisC_(T) is 86%, while the specificity is 86%.

The determination of the cutoff C_(T) of 36.255 is shown in Table 3. Theresults listed in Table 3 show that a cutoff C_(T) of 36.255 providedthe highest sensitivity and specificity.

The accuracy of the test depends on how well the test separates thegroup being tested into those with and without the prostate cancer.Accuracy is measured by the area under the ROC curve. Table 4 shows thecalculation of the area under the curve for the present example.

TABLE 3 Determination of Specificity and Sensitivity Positive if ≦^(a)Sensitivity 1 − specificity 19.86 .000 .000 24.87 .143 .000 29.48 .286.000 30.54 .429 .000 32.235 .429 .143 33.77 .571 .143 35.11 .714 .14336.255 .857 .143 37.415 .857 .286 39.23 .857 .429 39.995 1.000 .42940.21 1.000 .857 41.42 1.000 1.000 ^(a)the smallest cutoff value is theminimum observed test value minus 1 and the largest cutoff value is themaximum observed test value plus 1. All the other cutoff values are theaverages of two consecutive ordered observed test values.

TABLE 4 Results Showing Area Under the ROC Curve Asymptotic 95%Confidence Interval Area Std. Error^(a) Asymptotic Sig.^(b) Lower boundUpper bound .878 .096 .018 .689 1.066 Notes: ^(a)under thenon-parametric assumption ^(b)null hypothesis: true area = 0.5

Example 2 Association of Breast Cancer with 4 kb Deletion in Human mtDNA

Twenty breast tissue samples were collected, ten of which were malignantand ten of which had benign breast disease or no abnormalities. Thesesamples were formalin-fixed paraffin embedded and 20 micron sections ofeach were cut into individual sample tubes for extraction according tothe manufacturer's protocol for the QiaAMP DNA Mini Kit (Qiagen P/N51304). DNA was then quantified using a Nanodrop ND-1000 and normalizedto a concentration of 2 ng/ul.

Samples were then assayed for the levels of the 4 kb deletion byquantitative real-time PCR using the following protocol:

X iQ SYBR Green Supermix (Bio-Rad product no. 170-8880)

175 nmol forward primer (5′-TTGGTGCAACTCCAAAGCCACCCCTCACC-3′) (SEQ IDNO: 4)

175 nmol reverse primer (5′-AGGATGGTGGTCAAGGGAC-3′) (SEQ ID NO: 5)

20 ng template DNA in a 25 ul reaction

Reactions were cycled on an Opticon 2 DNA Engine (Bio-Rad Canada)according to the following protocol:

-   -   1.95° C. for 3 minutes    -   2. 95° C. for 30 seconds    -   3. 70° C. for 30 seconds    -   4. 72° C. for 30 seconds    -   5. Plate Read    -   6. Repeat steps 2-5 44 times    -   7. 72° C. for 10 minutes    -   8. Melting Curve from 50° C. to 105° C., read every 1° C., hold        for 3 seconds    -   9. 10° C. Hold

Tables 5 and 6, and FIG. 3 show the difference in the mean C_(T) scoresfor breast tissue samples from subjects having malignant breast tissueand benign breast tissue at the 0.065 level.

TABLE 5 Mean Values for C_(T) scores: Breast Tissue Samples Group N MeanStd. Dev. Std. Error Mean Normal 9 21.5278 2.71939 .90646 Malignant 918.9089 2.89126 .96375

TABLE 6 Significance Test for Mean C_(T) scores Test for Equality MeansLevene's 95% Test for Confidence Equality of Sig. Std. Interval of theVariances (2- Mean Error Difference CTt40 fluid F Sig. t df tailed)Diff. Diff. Lower Upper Equal .007 .934 1.979 16 .065 2.61889 1.32306−.18588 5.42366 variances assumed Equal 1.979 15.94 .065 2.61889 1.32306−.18674 5.42452 variances not assumed

Tables 7 and 8, and FIG. 4 illustrate that when using a cut-off cyclethreshold of 19.845 the sensitivity of the assay for breast cancer is78% and the specificity is 78%.

FIG. 4 is an ROC curve illustrating the specificity and sensitivity ofthe 4 kb mtDNA deletion as a marker for breast cancer when testingbreast tissue. These results were obtained using a cutoff C_(T) of19.845. The sensitivity of the marker at this C_(T) is 78%, while thespecificity is 78%.

The determination of the cutoff C_(T) of 19.845 is shown in Table 7. Theresults listed in Table 7 show that a cutoff C_(T) of 19.845 providedthe highest sensitivity and specificity.

The accuracy of the test depends on how well the test separates thegroup being tested into those with and without the breast cancer.Accuracy is measured by the area under the ROC curve. Table 8 shows thecalculation of the area under the curve for the present example.

TABLE 7 Determination of Specificity and Sensitivity Positive if ≦^(a)Sensitivity 1 − specificity 15.28 .000 .000 16.305 .111 .000 16.69 .222.000 17.075 .333 .000 17.4 .444 .000 17.71 .556 .000 18.0 .556 .11118.835 .556 .222 19.415 .667 .222 19.845 .778 .222 20.475 .778 .33310.79 .778 .444 21.38 .778 .556 22.005 .778 .667 23.145 .889 .667 24.19.889 .778 24.49 .889 .889 25.21 1.00 .889 26.66 1.00 1.00 ^(a)thesmallest cutoff value is the minimum observed test value minus 1 and thelargest cutoff value is the maximum observed test value plus 1. All theother cutoff values are the averages of two consecutive ordered observedtest values.

TABLE 8 Results Showing Area Under the ROC Curve Asymptotic 95%Confidence Interval Area Std. Error^(a) Asymptotic Sig.^(b) Lower boundUpper bound .778 .117 .047 .548 1.008

Example 3 Association of Prostate Cancer with 4 kb Deletion in HumanmtDNA Using Needle Biopsy Samples

Prostate needle biopsy specimens were obtained from 19 individuals, 9without prostate cancer and 10 with prostate cancer. Needle biopsytissues were formalin-fixed paraffin embedded (FFPE) as is standard inthe clinical diagnostic setting. 10 micron sections of each biopsy weredeposited directly into centrifuge tubes and the DNA was extracted usingthe QiaAMP DNA Mini Kit (Qiagen, p/n 51306). DNA extracts werequantified by absorbance at 260 nm using a NanoDrop ND-1000Spectrophotometer. Yields ranged from 347 ng to 750 ng. These sampleswere diluted to 2 ng/ul and amplification reactions setup according toTable 9 and the following:

TABLE 9 Reagents and Concentrations for  Amplification ReactionFinal Con- Reagent centration iQ SYBR Green Supermix   1 X(Bio-Rad Laboratories, p/n 170-8882) Forward Primer  175 nmol12303-12316/16243-16259F 5′- CCCAAAAATTTTGGTGCAACTCCAAAGCCAC -3′(SEQ ID NO: 6) Reverse Primer 16410R 175 nmol5′- AGGATGGTGGTCAAGGGAC -3′ (SEQ ID NO: 5) DNA extract 0.8 ng/u1

Nuclease-free water was added to a final reaction volume of 25 ul.Amplifications were carried out on a DNA Engine Chromo4 Real Time PCRInstrument (Bio-Rad Laboratories) according the following cyclingconditions:

1) 95° C. for 3 minutes

2) Followed by 45 cycles of

3) 95° C. for 30 seconds

4) 69° C. for 30 seconds

5) 72° C. for 30 seconds

6) Plate Read

Then

7) 72° C. for 10 minutes

8) Melting Curve 50° C.-105° C. reading every 1° C., hold for 3 seconds

9) 4° C. Hold

Results, shown in Table 10, demonstrate that those individuals withprostate cancer have a lower C_(T) value and therefore higher levels ofthe 4 kb deletion in prostate tissue than do those without prostatecancer. Patients with prostate cancer have an average C_(T) value of30.7 while the patients without prostate cancer have an average C_(T)value of 36.4. This difference of 5.7 C_(T) corresponds to nearly 100fold greater 4 kb deletion levels in the group with prostate malignancythan in the group without.

TABLE 10 Patient Diagnosis and Associated C_(T) Score Patient Number andDiagnosis C(t) CUG 1301 Malignant 25.7 CUG 1268 Malignant 27.7 CUG RN345 Normal 28.3 CUG 1272 Malignant 28.8 CUG 1375 Malignant 29.1 CUG 1259Malignant 29.1 CUG 1381 Malignant 30.2 CUG RN 82 Normal 30.5 CUG 1372Malignant 30.9 CUG 1085 C T1 Normal 31.5 CUG 1317 Malignant 31.7 CUG1377 F Normal 33.6 CUG 1365 B Normal 34.6 CUG 1370 Malignant 35.9 CUG RN405 Normal 37.5 CUG 1366 Malignant 37.9 CUG RN 701 Normal 41.7 CUG RN420 Normal 45 CUG RN 373 Normal 45

Tables 11 and 12 show the difference in the mean C_(T) scores forprostate tissue samples from subjects having normal and malignantprostate tissue.

TABLE 11 Mean Values for C_(T) Score: Prostate Needle Biopsy TissueGroup N Mean Std. Dev. Std. Error Mean Normal 9 36.4111 6.25229 2.08410Malignant 10 30.7 3.69534 1.16857

TABLE 12 Significance Test for C_(T) Scores Test for Equality MeansLevene's 95% Test for Confidence Equality of Sig. Std. Interval of theVariances (2- Mean Error Difference CTt40 fluid F Sig. t df tailed)Diff. Diff. Lower Upper Equal 4.426 .051 2.455 17 .025 5.71111 2.32589.80391 10.61831 variances assumed Equal 2.390 12.705 .033 5.711112.38935 .53701 10.88522 variances not assumed

Table 13 and FIG. 6 illustrate that when using a cutoff of C_(T) 32.65the sensitivity and specificity of correctly diagnosing these patientsis 80% and 67% respectively.

TABLE 13 Determination of Specificity and Sensitivity Positive if ≦^(a)Sensitivity 1 − specificity 24.7 .000 .000 26.7 .100 .000 28.0 .200 .00028.55 .200 .111 28.95 .300 .111 29.65 .500 .111 30.35 .600 .111 30.7.600 .222 31.2 .700 .222 31.6 .700 .333 32.65 .800 .333 34.1 .800 .44432.25 .800 .556 36.7 .900 .556 37.7 .900 .667 39.8 1.000 .667 43.351.000 .778 46.0 1.000 1.000

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto. All suchmodifications as would be apparent to one skilled in the art areintended to be included within the scope of the following claims. Alldocuments recited in the present application are incorporated herein byreference.

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1. A method of detecting a cancer in an individual comprising: a)obtaining a biological sample from the individual; b) extractingmitochondrial DNA (mtDNA) from the sample; c) quantifying the amount ofmtDNA in the sample having a deletion in the mtDNA sequence betweenabout residue 12317 and about residue 16254 of the human mtDNA genome;and d) comparing the amount of mtDNA in the sample having the deletionto at least one known reference value.
 2. The method of claim 1 whereinthe deletion has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2.
 3. The method of claim 1 wherein the at least one known referencevalue is the amount of the deletion in a reference sample of mtDNA fromknown non-cancerous tissue or body fluid.
 4. The method of claim 3wherein an elevated amount of the deletion in the biological samplecompared to the reference sample is indicative of cancer.
 5. The methodof claim 4 further comprising the step of comparing the amount of mtDNAin the sample having the deletion to the amount of the deletion in areference sample of mtDNA from known cancerous tissue or body fluid. 6.The method of claim 1 wherein the at least one known reference value isthe amount of the deletion in a reference sample of mtDNA from knowncancerous tissue or body fluid.
 7. The method of claim 6 wherein asimilar level of the deletion in the biological sample compared to thereference sample is indicative of cancer.
 8. The method of claim 7further comprising the step of comparing the amount of mtDNA in thesample having the deletion to the amount of the deletion in a referencesample of mtDNA from known non-cancerous tissue or body fluid.
 9. Themethod of claim 1 wherein the step of quantifying is conducted usingreal-time PCR.
 10. The method of claim 1 wherein the quantifying of thedeletion includes first amplifying a target region of mtDNA that isindicative of the deletion, and quantifying the amount of the amplifiedtarget region.
 11. The method of claim 10 wherein a PCR primer having asequence corresponding to SEQ ID NO: 4 is used as part of a pair ofamplification primers for amplifying the target region.
 12. The methodof claim 1 wherein the cancer is prostate cancer.
 13. The method ofclaim 1 wherein the cancer is breast cancer.
 14. The method of claim 1wherein the biological sample is a body tissue or body fluid.
 15. Themethod of claim 14 wherein the biological sample is selected from thegroup consisting of breast tissue, prostate tissue and urine.
 16. Themethod of claim 10 wherein the reference value is a cycle threshold. 17.A method of monitoring an individual for the development of a cancercomprising: a) obtaining a biological sample; b) extractingmitochondrial DNA (mtDNA) from the sample; c) quantifying the amount ofmtDNA in the sample having a deletion in the mtDNA sequence betweenabout residue 12317 and about residue 16254 of the human mtDNA genome;and d) repeating steps a) to c) over a duration of time; wherein anincreasing level of the deletion over the duration of time is indicativeof cancer.
 18. The method of claim 17 wherein the deletion has asequence as set forth in SEQ ID NO: 1 or SEQ ID NO:
 2. 19. The method ofclaim 17 further comprising at least one step selected from the groupconsisting of: (a) comparing the amount of mtDNA in the sample havingthe deletion to the amount of the deletion in a reference sample ofmtDNA from known non-cancerous tissue or body fluid; and (b) comparingthe amount of mtDNA in the sample having the deletion to the amount ofthe deletion in a reference sample of mtDNA from known cancerous tissueor body fluid.
 20. The method of claim 17 wherein the quantifying of thedeletion includes amplifying a target region of mtDNA that is indicativeof the deletion, and quantifying the amount of the amplified targetregion.
 21. The method of claim 20 wherein the step of quantifying isconducted using real-time PCR.
 22. The method of claim 20 wherein a PCRprimer having a sequence corresponding to SEQ ID NO: 2 is used as partof a pair of amplification primers for amplifying the target region. 23.The method of claim 17 wherein the cancer is prostate cancer.
 24. Themethod of claim 17 wherein the cancer is breast cancer.
 25. The methodof claim 17 wherein the biological sample is a body tissue or bodyfluid.
 26. The method of claim 25 wherein the biological sample isselected from the group consisting of breast tissue, prostate tissue andurine.
 27. The method of claim 10 wherein the amplifying of the targetregion is conducted using a pair of amplification primers, one of thepair of amplification primers overlapping a splice joining regions onopposite ends of the deletion.
 28. A method of detecting a cancer in anindividual comprising: a) obtaining a biological sample from theindividual; b) extracting mitochondrial DNA (mtDNA) from the sample; c)quantifying the amount of mtDNA in the sample having a deletioncorresponding to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO:2; and d) comparing the amount of mtDNA in the sample corresponding toSEQ ID NO: 1 or SEQ ID NO: 2 to at least one known reference value. 29.The method of claim 28 wherein the at least one known reference value isthe amount of the sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2in a reference sample of mtDNA from known non-cancerous tissue or bodyfluid.
 30. The method of claim 28 wherein the at least one knownreference value is the amount of the sequence corresponding to SEQ IDNO: 1 or SEQ ID NO: 2 in a reference sample of mtDNA from knowncancerous tissue or body fluid.
 31. The method of claim 28 wherein thestep of quantifying is conducted using real-time PCR.
 32. The method ofclaim 31 wherein the quantifying of the deletion includes firstamplifying a target region of mtDNA that is indicative of the deletion,and quantifying the amount of the amplified target region.
 33. Themethod of claim 32 wherein one of a pair of PCR primers used in theamplifying of the target region overlaps a rejoining site of thesequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2, after thesequence has re-circularized.
 34. The method of claim 28 wherein thecancer is prostate cancer.
 35. The method of claim 28 wherein the canceris breast cancer.
 36. The method of claim 28 wherein the biologicalsample is a body tissue or body fluid.
 37. The method of claim 36wherein the biological sample is selected from the group consisting ofbreast tissue, prostate tissue and urine.
 38. The method of claim 32wherein the reference value is a cycle threshold.
 39. A diagnostic kitfor carrying out the method of claim 1 comprising: (a) material forcollecting one or more biological samples; and (b) suitable primers andreagents for detecting the mtDNA deletion.